EP2518139A1 - Utilisation de dérivés d'indirubines pour la production des cellules souches pluripotentes - Google Patents

Utilisation de dérivés d'indirubines pour la production des cellules souches pluripotentes Download PDF

Info

Publication number
EP2518139A1
EP2518139A1 EP11003454A EP11003454A EP2518139A1 EP 2518139 A1 EP2518139 A1 EP 2518139A1 EP 11003454 A EP11003454 A EP 11003454A EP 11003454 A EP11003454 A EP 11003454A EP 2518139 A1 EP2518139 A1 EP 2518139A1
Authority
EP
European Patent Office
Prior art keywords
cells
indirubin derivative
group
smad1
pluripotent stem
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11003454A
Other languages
German (de)
English (en)
Inventor
Stefan Prof. Dr. Wölfl
Xintai Dr. Cheng
Hamed Alborzinia
Gerhard Prof. Dr. Eisenbrand
Karl-Heinz Dr. Merz
Ralf Prof. Dr. Mrowka
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universitaetsklinikum Jena
Original Assignee
Universitaetsklinikum Jena
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universitaetsklinikum Jena filed Critical Universitaetsklinikum Jena
Priority to EP11003454A priority Critical patent/EP2518139A1/fr
Publication of EP2518139A1 publication Critical patent/EP2518139A1/fr
Withdrawn legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/32—Oxygen atoms
    • C07D209/36—Oxygen atoms in position 3, e.g. adrenochrome
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/40—Nitrogen atoms, not forming part of a nitro radical, e.g. isatin semicarbazone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the present invention relates to a method for the production of pluripotent stem cells, comprising the step of incubating isolated mammalian cells with an indirubin derivative, the use of an indirubin derivative inducing isolated mammalian cells to become pluripotent stem cells, and an indirubin derivative for use in inducing mammalian cells in a patient to become pluripotent stem cells.
  • iPS induced pluripotent stem cell lines
  • the regulation of gene expression covers a series of complex biochemical mechanisms and one can categorize it broadly into three levels: 1) control of transcription, by epigenetic chromatin activation followed by utilization of cis- regulatory elements, including promoters, enhancers, silencers, or locus control elements; 2) splicing of precursors of messenger RNAs (pre-mRNAs) and subsequent mRNA nuclear-to-cytoplasmic transport, by splicing-specific elements and transport factors; and 3) post-transcriptional control, by affecting the translational efficiency, the sub-cellular localization or the stability of mRNA, followed by posttranslational events, e.g., protein modification and degradation. While at each level a series of distinct biochemical machineries is involved in controlling gene expression, these regulatory circuits bear signs of interactions.
  • the first level directs whether a gene is transcribed and to what extent.
  • chromatin around promoters and enhancers must be activated and the promoter region upstream in proximity to the transcription start site (TSS) is essential and contains sufficient information of transcription factor binding sites for the recruitment of the protein complex for RNA synthesis to direct the correct expression of a gene either alone or in conjunction with enhancer sequences.
  • Stem cells in particular embryonic stem cells, which can be expanded indefinitely and are pluripotent, have attracted considerable attention as a therapeutic approach for treating for example diabetes, cardiovascular-, neurological-, and liver-based diseases.
  • embryonic stem cells in particular embryonic stem cells, which can be expanded indefinitely and are pluripotent, have attracted considerable attention as a therapeutic approach for treating for example diabetes, cardiovascular-, neurological-, and liver-based diseases.
  • inner cell mass-derived embryonic stem cells in cell replacement therapy remains problematic for a number of reasons, including host rejection of allogeneic cells.
  • human ESCs are also associated with ethical issues regarding the use of human embryos.
  • iPS induced pluripotent stem
  • TGFß and BMP belong to the cytokine growth factor family and are the key regulators of embryonic development and the postnatal homeostasis of different organs by regulating cellular differentiation, extracellular matrix remodeling and proliferation ( Hong cytokine & growth factor review 2009 20 409-418 ).
  • the dysregulation of TGFß/BMP signalling is associated with several diseases, like cancer.
  • TGF-ß/BMP signaling is initiated by engaging of ligands to their corresponding receptors kinases type I and type II on cell surface leading to form a receptor heterocomplex which in turn phosphorylates Receptor-activated Smad1/5/8 (R-Smad) for BMP or R-Smad2/3 for TGFß at their C-termini of MH2 domain to provide a docking site for binding to the common partner Co-Smad4 facilitating R-Smad translocation into the nucleus, where upon accumulation the Smad complex binds to DNA with other elements to regulate target gene expression such as IDs ( Massague genes dev.
  • R-Smads comprise also an N-terminal MH1 domain connecting with MH2 domain via an interdomain termed linker region ( Eivers Cytokine & Growth factor reviews 2009 20 357-365 ).
  • linker region The phosphorylations of the linker region by various kinases play a crucial role in regulation of Smad-dependent TGFß/BMP activity ( Pera genes dev. 2003 3023-3028 ).
  • CML Chronic Myelogenous Leukemia
  • Indirubin and indirubin derivatives demonstrate the ability to inhibit protein kinases in ATP-pocket including serine/threonine kinases CDKs ( Hossel 1999 Stahl cell biol ), GSK-3ß ( Meijer 2003 chemistry & biology 10 1255-1266 ), tyrosine kinases c-Src ( Nam 2005 PNAS ) and FGFR1 ( Zhen oncogene 2007 26 6372-6385 E231 ) in a structure- and substituent-dependent manner ( Cheng 2010 Bioorg. Med. Chem. 18 4509-4515 ).
  • 6-bromoindirubin-3'-oximether BIO
  • IRDs 6-bromoindirubin-3'-oximether
  • the technical problem underlying the present invention is to provide new means for inducing pluripotency in differentiated cells.
  • the present invention relates to a method for the production of pluripotent stem cells, comprising the steps of
  • R 1 and R 2 in formulae (1) can be the same or different as below described:
  • the physiologically compatible salts of organic and inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, succinic acid, and others, are suitable, representing highly water soluble compounds. Transformation of the amino compounds into these salts is easily to be performed by chemists skilled in the art.
  • the indirubin derivative is selected from the group consisting of E739, E852, E862, E849, E823, E820, E861, E673, E692, E721, E740, E804, E738, E231, E860a, E748a, and E728 as shown in Figure 11 of the present application.
  • the indirubin derivative is selected from the group consisting of E231, E723, E728, E738, E748a, E804, and E860a, as shown in Figure 11 and Table 1 of the present application.
  • the indirubin derivative is E738 as shown in Figure 11 of the present application.
  • the term "mammalian cells” means a generic term and encompass the cultivation of individual cells, tissues, organs, primary cells, continuous cell lines, and/or genetically engineered cells, such as recombinant cells expressing a heterologous polypeptide or protein.
  • the mammalian cell is a differentiated cell, more preferably a differentiated adult cell.
  • the mammalian cell is a tumor cell or a tumor cell line.
  • the mammalian cell is derived from a neonatal, child, or adult.
  • the mammalian cell is not derived from an embryo.
  • the mammalian cell as used in the present invention is not an embryonic stem cell.
  • Mammalian cells suitable for being induced to pluripotent stem cells via incubation with an indirubin derivative the present according to the invention include those of human origin, which may be primary cells derived from a tissue sample, diploid cell strains, transformed cells or established cell lines. Mammalian cells can include human and non-human cells alike. Mammalian cells of non-human origin can be monkey cells, bovine cells, dog cells, pig cells, rabbit cells, mouse cells, rat cells, sheep cells, hamster cells, Chinese hamster ovarian cells or an animal cell derived from any tissue.
  • mammalian cells can be HeLa cells, C2C12 cells, NIH 3T3 cells, HFF cells, BSC-1 cells, LLC-MK cells, CV-1 cells, COS-cells, COS-1 cells, COS-3 cells, COS-7 cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK-cells, TCMK-1 cells, LLC-PK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK-21 cells, CHO cells, NS-1 cells MRC-5 cells, WI-38 cells, BHK cells, 293 cells and RK-cells.
  • the mammalian cells are selected from the group consisting of Human skin fibroblasts, Human mesenchymal stem cells (from blood), other differentiated human cells from biopsies, HFF cells, HeLa cells, mouse fibroblasts, C2C12 cells, NIH 3T3 cells.
  • the mammalian cells as used herein may be cultured cells that replicate and are capable of growing in cell culture or large scale cultivation in bioreactor.
  • the unrestricted growth of cultured cells permits long-term cultivation from a standardized cell substrate and low costs.
  • Mammalian cell lines can be selected from the group HeLa cells, C2C12 cells, NIH 3T3 cells and other cell lines.
  • the cultured cells are preferably tested for absence of adventitious agents, such as bacteria, fungi, mycoplasma, protozoans and viruses.
  • cell culture in its various grammatical forms, refers to cells grown in suspension, roller bottles, flasks and the like. Large scale approaches, such as bioreactors, including adherent cells growing attached to microcarriers in stirred fermentors, also are included. Moreover, it is possible to not only culture surface-dependent cells, but also to use the suspension culture techniques. If the cells are grown on microcarriers, the microcarrier can be selected from the group of microcarriers based on dextran, collagen, plastic, gelatin and cellulose and others.
  • Mammalian cells are typically cultivated in a cell incubator at about 37°C, with the culture medium having an optimal pH in the range of about 6.8 to 7.6, preferably between 7.0 and 7.3.
  • Cells in batch culture might have a complete medium change about every 2 to 3 days, or more or less frequently, if required.
  • Cells in perfusion culture e.g. in bioreactor or fermenter
  • Cultivation approaches can include, depending on context and need, the sub-cultivation, passaging and propagation of the cells.
  • the cells can be cultivated in any suitable cell culture medium known in the art.
  • Mammalian cells may be derived from any tissue type, like for example, lung, gut, muscles, bones, bone marrow, epithelium, connective tissue, blood vessels, and/or nervous tissue.
  • the mammalian cells are present in a biopsy of one of the above tissues.
  • mammalian cells may be derived from any body fluid, like for example urine, blood, blood products, blood plasma, cereospinal fluid, ammniotic fluid, and/or lymph.
  • the isolated mammalian cells are isolated human cells.
  • incubating the cells of step (c) with the indirubin derivative is carried out with at least 0.1 ⁇ M, at least 0.5 ⁇ M, at least 1 ⁇ M, at least 2 ⁇ M, at least 3 ⁇ M, at least 4 ⁇ M, at least 5 ⁇ M, at least 6 ⁇ M, at least 8 ⁇ M, or at least 10 ⁇ M indirubin derivative.
  • incubating the cells of step (c) with the indirubin derivative is carried out with 0.1 ⁇ M to 10 ⁇ M, more preferable 1 ⁇ M to 10 ⁇ M, more preferable 2 ⁇ M to 10 ⁇ M, more preferable 3 ⁇ M to 10 ⁇ M, more preferable 4 ⁇ M to 10 ⁇ M, more preferable 5 ⁇ M to 10 ⁇ M, more preferable 6 ⁇ M to 10 ⁇ M, and more preferable 8 ⁇ M to 10 ⁇ M indirubin derivative.
  • incubating the cells of step (c) with the indirubin derivative is carried out for at least 1 h, more preferable for at least 2h, more preferable for at least 3h, more preferable for at least 4h, more preferable for at least 6h, more preferable for at least 8h, more preferable for at least 10h, more preferable for at least 12h, more preferable for at least 18h, more preferable for at least 24h, more preferable for at least 48h, more preferable for at least 3 days, more preferable for at least 4 days, more preferable for at least 6 days, more preferable for at least 8 days, more preferable for at least 10 days, or more preferable for at least 12 days.
  • the cells of step (a) are incubated in medium supplemented with growth factors and/or FCS, preferably 10% (v/v) or 15% (v/v) FCS, and/or with MEF conditioned medium, and/or with HFF conditioned medium, and/or supplemented with BMP2, and/or supplemented with FGF2 either before step (c), preferably 1 hour before step (c), or simultaneously with the incubation with the indirubin derivative in step (c).
  • medium supplemented with growth factors and/or FCS preferably 10% (v/v) or 15% (v/v) FCS
  • MEF conditioned medium preferably 10% (v/v) or 15% (v/v) FCS
  • HFF conditioned medium preferably 1 hour before step (c)
  • FGF2 FGF2
  • the indirubin derivative activates genes that convey pluripotency, preferably in a reversible manner.
  • the indirubin derivative activates at least one gene selected from the group consisting of OCT4 , SOX2 , NANOG and LIN28 .
  • the present invention further relates to a use of an indirubin derivative for inducing isolated mammalian cells to become pluripotent stem cells.
  • the indirubin derivative and the isolated mammalian cells are as defined herein.
  • the use of an indirubin derivative for inducing isolated mammalian cells comprises carrying out the method according to the present invention.
  • the present invention relates to an indirubin derivative for use in inducing mammalian cells in a patient to become pluripotent stem cells.
  • the present invention relates to an indirubin derivative for use in inducing mammalian cells in a patient to become pluripotent stem cells, wherein the pluripotent stem cells regenerate a tissue in the patient.
  • the indirubin derivative and the isolated mammalian cells are as defined herein.
  • inducing mammalian cells in a patient to become pluripotent stem cells includes induction of any kind of cell or tissue in a patient to become a pluripotent stem cells.
  • patient does not underly any specific limitation as long as the patient is a mammal. In a preferred embodiment of the present invention the patient is a human.
  • Another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the above defined indirubin derivative for inducing mammalian cells in a patient to become pluripotent stem cells.
  • the indirubin derivative and the isolated mammalian cells are as defined herein.
  • the pharmaceutical composition may further comprise an auxiliary agent, e.g. selected from the group consisting of a pharmaceutically acceptable carrier, diluent, salt, buffer, or excepient. Said pharmaceutical composition can be used for treating the above-defined bleeding disorders. Further, the pharmaceutical composition may be administered by any route known in the prior art. In one example, the pharmaceutical composition may be administered intravenously, intraperitoneal, topically, or by site-specific injection.
  • an auxiliary agent e.g. selected from the group consisting of a pharmaceutically acceptable carrier, diluent, salt, buffer, or excepient.
  • Said pharmaceutical composition can be used for treating the above-defined bleeding disorders.
  • the pharmaceutical composition may be administered by any route known in the prior art. In one example, the pharmaceutical composition may be administered intravenously, intraperitoneal, topically, or by site-specific injection.
  • the indirubin derivative, the pharmaceutical composition and the mammalian cells in a patient to become pluripotent stem cells are as defined above.
  • the present invention further relates to a indirubin derivative E738 as shown in Figure 11 of the present application.
  • the invention underlying the present application identifies and optimizes small molecules that activate the endogenous expression of the genes OCT4 , SOX2 , NANOG , and LIN28 and transcriptional networks pertinent for maintaining self-renewal and pluripotency of cells derived from adult human tissue.
  • the present invention addresses a highly relevant issue of modern biology and medicine.
  • Pluripotent human stem cells are useful for multiple applications in transplantation medicine and tissue engineering as well as for the generation of new disease models and in drug development.
  • Induced pluripotent human stem cells that are derived from adult tissue may serve as a complementary alternative to inner cell mass (ICM)-derived embryonic pluripotent stem cells that are highly debated regarding ethical issues.
  • ICM inner cell mass
  • generation of pluripotent stem cells from an adult will open completely new possibilities since auto transplantation of engineered tissue will not be subjected to tissue rejection due to immune responses.
  • the present invention provides chemical small molecule compounds that would allow activating the mentioned human genes that convey pluripotency in a reversible manner. This approach makes it possible to use pluripotent human stem cells in therapeutic contexts and would allow translating that knowledge into products.
  • IRDs were obtained by Prof. Eisenbrand in Kaiserslautern, Germany. Structures and purities were ascertained by 13C- and 1 H-NMR spectroscopy and elemental analyses. Dorsomorphin and Repsox were purchased from Calbiochemie. MG132 was from Sigma-Aldrich (Germany).
  • p-Smad1/5/8 C-terminal
  • p-Smad1 S206
  • p-JNK JNK
  • p-P38 P38
  • p-Erk1/2 Erk1/2
  • p-Smad3 Smad2/3, p-Stat3, Stat3, Non-p-ß-catenin 41/37/33, Ubiquintin and ß-Actin antibody
  • Smad1/5/8 was from Santa Cruz (Germany).
  • the serum-depleted C2C12 cells were coinbubated with BMP2 (100ng/mL) and E738 at various concentration as indicated for 12h. Cells were fixed in 4% PFA and immunostained with Smad1/5/8 antibody (1:100) and DAPI as described previously.
  • the Kinase profiling assay was performed by ProQinase as previously described (Cheng et al.201 0).
  • IRDs intefere with BMP signaling in Hela cells and human fibroblasts
  • E738 decreases both BMP- and TGFß-signaling associated R-Smads after 24h treatment
  • BIO 6-Bromo-indirubin-3'-oxim
  • GSK3ß IC 50 : 5nM
  • CDK5/p25 83nM
  • BIO showed the benefits in maintenance of mouse and human ESCs and in generation of human ESC-like mouse epiblast Stem cell.
  • FCS stimulated cells demonstrated the activation of both types of R-Smads, which were repressed by indirubins, whereas only partly by DM and Repsox ( Fig. 2D lower).
  • indirubins repressed Smad-assiciated pathways more efficient than receptor specific inhibitors.
  • receptor specific inhibitors can only inactivate Smads by inhibition of limited target receptors.
  • Smad turnover-associated protein kinases are regulated by E738
  • E738 initially was designed as a protein kinase inhibitor, we asked if E738 induced dephoshorylation and degradation are caused by certain protein kinases.
  • protein kinase profiling containing several Smad-relative protein kinases like CDKs and GSK3ß to overview the inhibitory effect of E738 in vitro.
  • the residual activities of kinases were measured by using recombinant protein kinases and proper substrates after treatment with 1 ⁇ M E738 in the presence of 1 ⁇ M ATP as our previously described.
  • Zhu et al. reported that E231 can stimulate the phosphorylation of p38, which in turn activates Erk1/2. However the mechanism was still remained to elucidate.
  • Raf kinase inhibitor may induce the activation of MAPKs in vitro and in vivo leading to the failure of cancer therapy ( Hatzivassiliou nature 2010 464 431-436 , Heidol cell 2010 140 209-221 , Murphy PNAS 2010 107 9 4299-4304 ).
  • the indirubin activated MAPKs might be due to its inhibition against Raf kinase.
  • the Lin et. al. described Stat3 inhibitor could disrupt TGFß signaling ( Lin oncogene 200928 961-972 ). Therefore we included Raf and Stat3-relative protein kinases, like Jak, Kit and Src, in this small screening.
  • CDKs complexes like CDK1/CycB1, CDK2/CycA, CDK2/CycE, CDK4/CycD1 and CDK5/p35, were dramatically decreased to 16%, 10%, 8%, 23% and 16% respectively, as well as GSK3ß, a key modulator of Wnt signalling, its residual activity was remained only 6%.
  • Stat3 pathway associated kinases like JAK2, KIT and Src were also generally inhibited.
  • E738 blocked the 57% activity of b-Raf at this concentration ( Fig. 7 ).
  • the active p38 and JNK may hyper-phosphorylate p-Smad1/5/8 (C-Terminal) in linker region facilitating its binding to Smurf1 to regulate its turnover ( sapkota molecular cell 2007 25 441-454 ).
  • E738 promoted the phosphorylation of p38 and JNK.
  • MAPK inhibitors neither alter the level of pan Smad1/5/8 alone, nor attenuate the E738-mediated degradation in combination with E738. The finding implied that MAPKs might not be crucial in the indirubin-mediated degradation.
  • E738 is unable to immediately block the phosphorylation of Smad1/5/8 in response to BMP encourage us to hypothesize that E738 might not be BMP receptor inhibitor.
  • the p-Smad1/5/8 antibody detected an acute inhibitory effect over the period in the DM treatment and a rapid rescue recovery after removing DM ( Fig. 3D lower).
  • E7308 we found it stabilized p-Smad1/5/8 rather than inhibited in the first 3h of treatment, which is in agreement to its ability to inhibition against CDKs and GSK3ß to elevate the duration of p-Smad1/5/8. Indeed, the appearances of E738 repressed phosphorylation and degradation were observed simultaneously 5h after treatment, 2h later after the removal of compound. The results represented a delayed effect and evidenced a distinct behaviour on E738 induced inhibition in comparison to DM providing our hypothesis that E738 inhibited Smads activities in a receptor-independent manner.
  • DM as additive surprisingly promoted the degradation in combination with E738 even at 0.5 ⁇ M.
  • Enhanced degradation effect by coincubation with BMP receptor inhibitor and E738 provided our hypothesis that C-tail phosphorylation of Smads is not invoked in the degradation.
  • the results may imply certain proteins are regulated by DM, which also contribute to E738 induced degradation, or C tail phosphorylation of Smad1/5/8 may prevent the degradation due to translocation from cytosol to nuclei.
  • Ubiquitin-proteasome degradation system is the major route to protein degradation.
  • MG132 a general proteasome inhibitor, to characterize the role of proteasome in degradation.
  • 5 ⁇ M MG132 was sufficient to rescue the E738 mediated Smad1/5/8 ubiquitination 24h after treatment ( Fig. 5E and 5F ).
  • MG132 alone significantly stabilized mono-, di- and poly-ubiquitins ( Fig. 5F ).
  • more mono-ubiquitin, but less di- and poly-ubiquitins were observed in the presence of E738.
  • LiCl a GSK3ß inhibitor
  • BMP activated Smad1/5/8 but not change the level of pan Smad1/5/8 implying the specific interaction between Wnt and BMP signaling as recently reported.
  • Wnt signaling on BMP is not crucial in indirubin regulated BMP/Smad pathway ( Fig. 5F ).
  • E738 generally inhibited tumor cells growth with IC50-values below 1 ⁇ M and induced Parp cleavage, while its toxicities towards healthy cell lines were 20-fold lower.
  • E738 more efficiently (above 10-fold) inhibited the HeLa cells growth in response to BMP2 than FCS due to facilitate the ubiquitination of R-Smads, the key elements of BMP pathway ( Fig. 10B and 4C ).
  • IRDs were synthesized by Dr. Cheng, Dr. Merz and Prof. Eisenbrand in Kaiserslautern, Germany, (see Figure 11 ). Structures and purities were ascertained by 13 C- and 1 H-NMR spectroscopy and elemental analyses as reported previously ( Cheng, Dissertation, Kaiserslautern 2009 ; Eisenbrand, EP 08 022 288.8-2101, 2008 ).
  • HeLa and Hek293T were cultured in DMEM (PAA) containing 10% FCS (PAA) and 1% PS (PAA).
  • PAA DMEM
  • PAA DMEM
  • PAA DMEM
  • PS 1% PS
  • HFF human foreskin fibroblasts
  • Mouse embryonic fibroblasts were cultured in DMEM containing 10% NCS (LifeTechnologies) and 1% PS.
  • the cells were maintained under 5% CO2 at 37°C in a humidified atmosphere.
  • the cells were treated with various compounds as indicated in context.
  • the images were taken at indicated time points using a Keyence digital-microskop BZ-800.
  • Protein Extraction Western blot Cell extracts were homogenized in Urea-Lysis buffer (1mM EDTA, 0.5% Triton X-100, 5mM NaF, 6M Urea, 1mM Na 3 VO 4 , 10 ⁇ g/mL Pepstatin, 100 ⁇ M PMSF and 3 ⁇ g/mL Aprotinin in PBS). Enhanced chemiluminescence (ECL) Western blot analysis was performed. 20-40 ⁇ g of total protein extracts were resolved on 10% SDS-PAGE gels and immunoblotted with specific antibodies.
  • Urea-Lysis buffer 1mM EDTA, 0.5% Triton X-100, 5mM NaF, 6M Urea, 1mM Na 3 VO 4 , 10 ⁇ g/mL Pepstatin, 100 ⁇ M PMSF and 3 ⁇ g/mL Aprotinin in PBS.
  • ECL Enhanced chemiluminescence
  • P-Akt Primary antibodies
  • N-Smad3 and Oct3/4 were from Santa Cruz. Nanog was from Abcam.
  • Sox2 was from Invitrogen.
  • E-Cadherin was from Epitomics (Biomol, Germany)) were incubated at a 1:1,000 dilution in TBS (pH 7.5) with 0.1% Tween-20 and 5% BSA/nonfat milk (according to manufacture introduction) with gentle agitation overnight at 4°C.
  • iPS-Colony formation and spontaneous differentiation in HEK 10 5 cells/well Hek 293T were seeded in a 12-well plate in 10% FCS with 1mL/well). Next day, the cells were treated with E738 (8 ⁇ M). After 24h, a clear EMT could be observed. The floating cells were collected, washed with PBS and replated in 96-well plate in the presence of 10% FCS (200 ⁇ M/well) for 3 days. iPS-like colonies were observed. Afterwards, the cells were incubated further in the presence of 10% FCS and the medium was refreshed every 3 days. The spontaneous differentiation occurred after 10 days incubation.
  • iPS-Colony formation and Alkaline phosphatases staining in HFF 10 5 cells/well HFF at passage 4 were seeded in a 12-well plate in 15% FCS. Next day, the cells were treated with indicated concentrations of E738 without change of medium for indicated times. After the appearance of morphologic transition and stem cell like colony formation, alkaline phosphatase staining was performed using StemTAG Alkaline phosphatase staining kit (Biocat, Germany) according to the manufacturers protocol.

Landscapes

  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Transplantation (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
EP11003454A 2011-04-27 2011-04-27 Utilisation de dérivés d'indirubines pour la production des cellules souches pluripotentes Withdrawn EP2518139A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP11003454A EP2518139A1 (fr) 2011-04-27 2011-04-27 Utilisation de dérivés d'indirubines pour la production des cellules souches pluripotentes

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP11003454A EP2518139A1 (fr) 2011-04-27 2011-04-27 Utilisation de dérivés d'indirubines pour la production des cellules souches pluripotentes

Publications (1)

Publication Number Publication Date
EP2518139A1 true EP2518139A1 (fr) 2012-10-31

Family

ID=46085526

Family Applications (1)

Application Number Title Priority Date Filing Date
EP11003454A Withdrawn EP2518139A1 (fr) 2011-04-27 2011-04-27 Utilisation de dérivés d'indirubines pour la production des cellules souches pluripotentes

Country Status (1)

Country Link
EP (1) EP2518139A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103435606A (zh) * 2013-08-22 2013-12-11 中国药科大学 CDK2与GSK3β双重抑制剂及用途
JP2016514160A (ja) * 2013-03-14 2016-05-19 シティ・オブ・ホープＣｉｔｙ ｏｆ Ｈｏｐｅ ５−ブロモ−インジルビン
CN110536686A (zh) * 2017-04-18 2019-12-03 Ck生物技术公司 包含作为有效成分的靛玉红衍生物的药物组合物
US10703718B2 (en) 2014-03-14 2020-07-07 City Of Hope 5-bromo-indirubins
CN111542513A (zh) * 2017-10-31 2020-08-14 佩勒梅德有限公司 用于预防或治疗急性髓性白血病或转移性乳腺癌的药物组合物

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010033906A2 (fr) * 2008-09-19 2010-03-25 President And Fellows Of Harvard College Induction efficace de cellules souches pluripotentes au moyen de composés à petite molécule
WO2010068955A2 (fr) * 2008-12-13 2010-06-17 Dna Microarray Dosage de niche micro-environnementale pour criblage de cellules souches pluripotentes induites (cips)

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010033906A2 (fr) * 2008-09-19 2010-03-25 President And Fellows Of Harvard College Induction efficace de cellules souches pluripotentes au moyen de composés à petite molécule
WO2010068955A2 (fr) * 2008-12-13 2010-06-17 Dna Microarray Dosage de niche micro-environnementale pour criblage de cellules souches pluripotentes induites (cips)

Non-Patent Citations (30)

* Cited by examiner, † Cited by third party
Title
ABREU, NATURE CELL BIOLOGY, vol. 4, 2002, pages 599 - 607
ALARCON, CELL, vol. 139, 2009, pages 757 - 769
BENEZRA R., CELL, vol. 61, 1990, pages 49 - 59
CHENG, BIOORG. MED. CHEM., vol. 18, 2010, pages 4509 - 4515
DM, YU, NAT CHEM BIOL, vol. 4, 2008, pages 33 - 41
EISENBRAND, J. CANCER RES CLIN ONCOL, vol. 130, 2004, pages 627 - 635
EIVERS, CYTOKINE & GROWTH FACTOR REVIEWS, vol. 20, 2009, pages 357 - 365
FUENTEALBA, CELL, vol. 131, no. 5, 2007, pages 980 - 993
GELLIBERT, J MED CHEM, vol. 47, 2004, pages 4494 - 4506
HATZIVASSILIOU, NATURE, vol. 464, 2010, pages 431 - 436
HEIDOL, CELL, vol. 140, 2010, pages 209 - 221
HONG, CYTOKINE & GROWTH FACTOR REVIEW, vol. 20, 2009, pages 409 - 418
HOSSEL, NATUR CELL BIOI, 1999
ICHIDA, CELL STEM CELL, vol. 5, 2009, pages 491 - 503
KAHLEM, DEVELOPMENT, vol. 136, 2009, pages 3729 - 3740
LI, CELL STEM CELL, vol. 4, 2009, pages 513 - 524
LIN, ONCOGENE, vol. 200928, pages 961 - 972
MASSAGUE, GENES DEV., vol. 19, 2005, pages 2783 - 2810
MEIJER, CHEMISTRY & BIOLOGY, vol. 10, 2003, pages 1255 - 1266
MURPHY, PNAS, vol. 107, no. 9, 2010, pages 4299 - 4304
NAM, PNAS, 2005
PERA, GENES DEV., 2003, pages 3023 - 3028
POT CURRENT MOLECULAR MEDICINE, vol. 8, 2008, pages 319 - 328
SAPKOTA, MOLECULAR CELL, vol. 25, 2007, pages 441 - 454
XU, CELL STEM CELL, vol. 3, 2008, pages 196 - 206
YU J, VODYANIK MA, SMUGA-OTTO K, ANTOSIEWICZ-BOURGET J, FRANE JL, TIAN S, NIE J, JONSDOTTIR GA, RUOTTI V, STEWART R, SLUKVIN: "Induced pluripotent stem cell lines derived from human somatic cells", SCIENCE, vol. 318, 2007, pages 1917 - 20
YU, NAT CHEM BIOL, vol. 4, 2008, pages 33 - 41
ZHEN, ONCOGENE, vol. 26, 2007, pages 6372 - 6385
ZHU ET AL., NATURE, 2006
ZHU, NATURE, vol. 400, 1999, pages 687 - 693

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11306072B2 (en) 2013-03-14 2022-04-19 City Of Hope 5-bromo-indirubins
JP2016514160A (ja) * 2013-03-14 2016-05-19 シティ・オブ・ホープＣｉｔｙ ｏｆ Ｈｏｐｅ ５−ブロモ−インジルビン
CN103435606A (zh) * 2013-08-22 2013-12-11 中国药科大学 CDK2与GSK3β双重抑制剂及用途
US10703718B2 (en) 2014-03-14 2020-07-07 City Of Hope 5-bromo-indirubins
US11964941B2 (en) 2014-03-14 2024-04-23 City Of Hope 5-Bromo-indirubins
CN110536686A (zh) * 2017-04-18 2019-12-03 Ck生物技术公司 包含作为有效成分的靛玉红衍生物的药物组合物
JP2020517743A (ja) * 2017-04-18 2020-06-18 シーケー・バイオテクノロジー・カンパニー 活性成分としてインジルビン誘導体を含有する医薬組成物
EP3613419A4 (fr) * 2017-04-18 2021-01-06 CK Biotechnology Co. Composition pharmaceutique contenant de l'indirubine en tant que substance active
CN110536686B (zh) * 2017-04-18 2023-03-24 Ck雷容股份有限公司 包含作为有效成分的靛玉红衍生物的药物组合物
US11459311B2 (en) 2017-04-18 2022-10-04 Ck Regeon Inc. Pharmaceutical composition containing indirubin derivative as active ingredient
EP3705474A4 (fr) * 2017-10-31 2021-06-09 Pelemed Co., Ltd. Composition pharmaceutique pour la prévention ou le traitement de la leucémie myéloïde aiguë ou du cancer du sein métastatique
RU2763346C2 (ru) * 2017-10-31 2021-12-28 Пелемед Ко., Лтд. Фармацевтическая композиция для предупреждения или лечения острого миелоидного лейкоза или метастатического рака молочной железы
US11370779B2 (en) 2017-10-31 2022-06-28 Pelemed Co., Ltd. Pharmaceutical composition for preventing or treating acute myeloid leukemia or metastatic breast cancer
AU2018358582B2 (en) * 2017-10-31 2021-06-10 Pelemed Co., Ltd. Pharmaceutical composition for prevention or treatment of acute myeloid leukemia or metastatic breast cancer
JP2021501208A (ja) * 2017-10-31 2021-01-14 ペレメッド カンパニー リミテッドＰｅｌｅｍｅｄ Ｃｏ．， Ｌｔｄ． 急性骨髄性白血病又は転移性乳癌の予防又は治療用薬剤学的組成物
CN111542513A (zh) * 2017-10-31 2020-08-14 佩勒梅德有限公司 用于预防或治疗急性髓性白血病或转移性乳腺癌的药物组合物

Similar Documents

Publication Publication Date Title
EP3031908B1 (fr) Méthode de production de neurones dopaminergiques
Braydich-Stolle et al. Role of Src family kinases and N-Myc in spermatogonial stem cell proliferation
Palorini et al. Energy Metabolism Characterization of a Novel Cancer Stem Cell‐L ike Line 3 AB‐OS
US10233426B2 (en) Method for inducing cardiac differentiation of pluripotent stem cell with low-molecular compounds
Twaroski et al. Down-regulation of microRNA-21 is involved in the propofol-induced neurotoxicity observed in human stem cell–derived neurons
Resende et al. Influence of spontaneous calcium events on cell-cycle progression in embryonal carcinoma and adult stem cells
EP2518139A1 (fr) Utilisation de dérivés d'indirubines pour la production des cellules souches pluripotentes
Onizuka et al. Wnt2 accelerates cardiac myocyte differentiation from ES-cell derived mesodermal cells via non-canonical pathway
EP3305889B1 (fr) Procédé permettant d'induire une différenciation des cellules de crête neuronale en cellules du système nerveux autonome
US10196609B2 (en) Composition for promoting cardiac differentiation of pluripotent stem cell comprising EGFR inhibitor
Müller et al. Ca2+ activated K channels-new tools to induce cardiac commitment from pluripotent stem cells in mice and men
CA3182115A1 (fr) Procedes de differenciation de cellules souches en cellules progenitrices dopaminergiques
Wang et al. Constitutive activity of a G protein-coupled receptor, DRD1, contributes to human cerebral organoid formation
Ryu et al. Zinc chloride stimulates DNA synthesis of mouse embryonic stem cells: involvement of PI3K/Akt, MAPKs, and mTOR
Choi et al. Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis
Harbom et al. The effect of rho kinase inhibition on morphological and electrophysiological maturity in iPSC-derived neurons
Hwang et al. Sestrin2 attenuates cellular senescence by inhibiting NADPH oxidase 4 expression
La Torre et al. The GIPC1-Akt1 pathway is required for the specification of the eye field in mouse embryonic stem cells
Wang et al. Neuregulin-1 enhances differentiation of cardiomyocytes from embryonic stem cells
BR112019027282A2 (pt) método para induzir a diferenciação em células neuronais sensoriais periféricas e método de triagem de um agente biológico in vitro
US20200062719A1 (en) Application of Imidazopyridine Derivatives in Regenerative Medicine
Sun et al. Involvement of hepatic stimulator substance in the regulation of hepatoblast maturation into hepatocytes in vitro
WO2017188082A1 (fr) Additif pour milieu
Lan et al. Musashi 1-positive cells derived from mouse embryonic stem cells treated with LY294002 are prone to differentiate into intestinal epithelial-like tissues
US20220204925A1 (en) Generation of norepinephrine neurons from human stem cells

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

RIN1 Information on inventor provided before grant (corrected)

Inventor name: MERZ, KARL-HEINZ, DR.

Inventor name: CHENG, XINLAI, DR.

Inventor name: MROWKA, RALF, PROF. DR.

Inventor name: EISENBRAND, GERHARD, PROF. DR.

Inventor name: ALBORZINIA, HAMED

Inventor name: WOELFL, STEFAN, PROF. DR.

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20130503